The discovery that regulatory RNAs control biological pathways have revolutionized our understanding of gene expression over the past decade. At the forefront, microRNAs (miRNAs) have proven to be an abundant and essential class of RNA molecules in plants and animals. The importance of miRNAs in human biology is highlighted by the new recognition that mis-regulation of specific miRNA pathways underlies complex diseases, including cancer, heart ailments and neuronal pathologies. To understand the role of miRNAs under normal and disease conditions, two basic questions must be addressed: (1) how is miRNA expression regulated and how is this altered in the disease state, and (2) what are the biologically relevant miRNA targets, whose mis-regulation causes the disease phenotypes? These problems are the foundation of the work proposed here to elucidate a novel mechanism for controlling miRNA biogenesis and to determine how the miRNA complex recognizes and regulates specific targets in vivo. Typically, miRNAs recruit Argonaute (AGO) and its co-factors to specific mRNAs to trigger decay or translational repression. Here, an entirely new role for AGO in regulating miRNA biogenesis will be investigated. In Aim 1, the mechanism used by AGO to enhance the processing of let-7, and potentially other miRNAs, will be determined in C. elegans and human cells. These studies will reveal a novel pathway for controlling miRNA expression that may be broadly relevant for understanding how miRNA levels fluctuate during development and disease progression. Aim 2 will tackle the daunting problem of how imperfect base-pairing suffices for the regulation of specific targets by miRNAs. Three complementary methods will be employed to decipher how AGO recognizes and regulates specific targets in the endogenous context. These strategies take advantage of sensitive biochemical methods, unique worm strains and robust computational pipelines to assess the genome wide targeting properties of AGO guided by specific miRNAs in C. elegans. The consequences of AGO binding to its targets will also be analyzed to provide a comprehensive view of miRNA function in a live animal. Ultimately, these studies will yield unprecedented datasets for deciphering the rules used in vivo for miRNA target recognition and regulation in a multicellular animal.